The Effect Of Earthworm Digestion On The Abundance Of Tetracycline Resistance Genes In Sludge

As an environment-friendly and efficient technology,composting of waste from breeding industry and sewage sludge by earthworms,which can accelerates the conversion of waste to bio-fertilizer,plays a very important role in relieving the increasing environmental pressure from living and productive organic waste.Antibiotic resistance genes(ARGs),which is a new pollutant caused by overuse of antibiotics,have been attracted extensive attention by the academic circle.Waste from sewage treatment plant or livestock and poultry farm contain a large number of ARGs,which are important sources of environmental diffusion in farmland soil with ARGs.Earthworms play an important role in soil ecosystem and composting system.Therefore,studying the mechanism of earthworm digestion on antibiotic resistance genes has great environmental and ecological significance.In this paper,we studied the evolution of the bacterial community of the earthworm gut and womicast during earthworm digestion of cow dung and sludge sewage by the Illumina MiSeq sequencing and real-time qPCR,and discussed the variation law and mechanism of the abundance of tetracycline resistance gene(TRGs)in gut.The main results obtained in this paper are as follows:1.The characteristics of bacterial community structure in earthworm foregut,earthworm hindgut and mature wormcast were analyzed under the feeding of different baits(CD,cow dung and DS,sludge).The results show that:(1)Results showed the diversity and richness of bacterial community in earthworm gut significantly lower than that of baits,but the richness and diversity of wormcast bacterial community increased significantly after ripening.And the bacterial comlunity between CD and DS showed great differences,but the difference decreases and converges after earthworm digestion.(2)Proteobacteria,Bacteroides,Actinomycetes,Chloroflexi and Firmicutes were the dominant bacteria phyla in earthworm gut and wormcast,and Proteobacteria is the most abundant and an important component of earthworm composting.(3)The relative abundance of ?-proteobacteria in CD and DS accounted for 9.45%and 35.79%respectively,increased to 73.02%and 85.43%in foregut,decreased gradually in hindgut and decreased to 6.44%and 8.74%in wormcast.The relative abundance of ?-proteobacteria in CD and DS accounts for 13.7%and 6.45%respectively,and died rapidly in foregut,but recovers in the hindgut,and further augment in wormcast.The relative abundance of a-proteobacteria accounts for about 2.5%of CD and DS respectively,display different degrees of expansion in earthworm gut and decreas in wormcast,indicating that ?-proteobacteria mainly expands in gut..(4)Although the bacterial community structure of bait(CD and DS)varied greatly,the difference decreased and converged after digestion by earthworms.We also found Amycolatopsis,Methylobacterium,Ralstonia,Ochrobactrum and Sphingomonas are common in earthworm gut.2.The bacterial community structure of earthworm gut and wormcast under different TC residual levels was studied under the feeding of sludge.,the results show that:(1)the increase of TC residual level significantly increased the richness and diversity of bacteria community in foregut,and the diversity and richness of bacteria community in hindgut were significantly lower than that of the control under medium and low TC concentrations,while the opposite trend was observed under high TC concentrations.In addition,the richness and diversity of bacteria community in wormcast under different TC residual levels were significantly lower than those of control.(2)TC has inhibitory effect on the Proteobacteria and ?-Proteobacteria in gut bacterial community,but has different promoting effect on Actinobacteria,Chloroflexi,Firmicutes,Planctomycetes,Acidobacteria,?-proteobacteri,Anaerolmeae,?-proteobacteria,Actinobacteria,Acidimicrobia,Thermomicrobia and Thermoleophilia.The changes in gut(especially in foregut)bacterial community were affected by TC stress,which may afreet the efficiency of decomposing and transforming the bait.However,the bacterial community of mature wormcast is no significant difference under different levels of TC residues,which indicates that the wormcast ripening can eliminate the effect of TC residues on gut bacterial community.3.The abundance of TRGs(tetB,tetC,tetM,tetO,tetT,tetZ,tetL and tetX)and intl1 in the gut were studied by real-time qPCR under different TC concentrations.Results showed that:(1)the abundance of most TRGs and intl1 in the gut system was significantly lower than that of DS under the TC concentration of 0?25mg/kg.TC has inhibitory effect on the abundance of TRGs when the concentration is 5 mg/kg,and no obvious effect on the abundance of TRGs when the concentration is 25 mg/kg,but obviously promoted the abundance of TRGs and intl1 in gut(especially in hindgut)when the concentration is 125 mg/kg.Moreover,the abundance of tetB,tetZ,tetM and tetT were significantly higher than DS in the background of high concentration.These results indicated controlling the content of TC in sludge is a great significance for the control of the TRGs during the composting process.(2)In addition,we performed a redundant analysis between 15 dominant species and TRGs in earthworm gut,and found that the dominant taxon in earthworm gut are sensitive to TC,and the dominant taxon with inhibited in foregut was restored because of the abundance ot TRGs reduced in the hindgut.The correlation between TRGs and the dominant taxon in the hindgut is stronger the in the foregut,implying the bacterial hosts with carrying multiple TRGs are more abundant in the hindgut than in the foregut.(3)Correlation analysis shows that 18 genera may be the TRGs hosts in gut,and among which 5 unique genera in the foregut include the common genera of gut,and the genera from sludge.Calikinea,Unclassified_JG30-KF-CM45,Unclassified_Actinomycetales,Unclassifled_Acidimicrobiales,and Unclassified_HOC36 are the common bacteria genera in gut.Moreover,Unclassified_Actinomycetales and Acidimicrobiales are the main genera in gut,while Caldilinea comes from bait.The three genera with carrying abundant TRB in the hindgut are not appear in the top 15 abundant genera,among which Thermononas and Unclassified Chitinophagaceae are abundant in earthworm compost or earthworm gut.These genera may cause a diffusion risk of TRGs due to the abundance substantial increase in the hindgut.